X-Gal

X-Gal is a chromogenic substrate of ß-ga-lactosidase. X-Gal is used in conjunction with Isopropyl-b-D-1-thiogalactoside (IPTG) (I1401) for the detection of ß-galactosidase activity in bacterial colo-nies in a colorimetric assay in order to distinguish recombinants (white) from non-recombinants (blue).X-gal is cleaved at the ß1-4 bond between galacto-se and the 5-Bromo-4-chloro-3-indolyl part of X-Gal by ß-galactosidase via hydrolysis. The enzymatic cleavage of X-Gal results in the pro-duction of a water insoluble blue dichloro-dibromo-indigo precipitate. In cloning strategies with vectors like Lamb-da-11, M-13m-p18 and 19, pUC18 and 19, pUR222 the E. coli lacZ gene is transformed to lac- cells. After transfor-mation, the cells show ß-galactosidase activity in the presence of IPTG and X-Gal containing media. The insertion of a DNA frag-ment into the cloning sites of the lacZ gene results in the disruption of ß-galactosidase activity leading to the appearance of white colonies on X-Gal and IPTG containing media. Non recombinant cells produce a blue indigo dye on these media